Micropropagation from Cotyledonary Explants of Grapefruit (citrus Paradisi Macf.) by Indirect and Direct Organogenesis

Grapefruit (Citrus paradisi Macf.) is an economic fruit crop worldwide cultivated for its nutritional value, and aromatic flavours. However, the development of new varieties and high production of quality seedling constitutes a limit to improving grapefruit yields. An efficient regeneration protocol could be of great interest for grapefruit transformation, micropropagation, and germplasm conservation. In vitro plant regeneration systems of Citrus paradisi have been studied by using cotyledonary explants. Different concentrations of plant growth regulators were used to explore the organogenic potential of cotyledonary explants under laboratory conditions. For indirect organogenesis using cotyledon segment explants, a low concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) lead to the formation of white callus, while a high concentration of 2,4-D induced brown callus and adventitious root differentiation. The best response for callogenesis was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg/L of 2,4-D. Only brown callus cultured on MS added with 2 mg/L 6-benzylaminopurine (BAP) has regenerated shoots, but the percentage of callus producing shoots was very low (6.66 ± 2.68%). Regarding direct organogenesis from the cotyledonary node explants, the best response for shoot proliferation was observed on MS supplemented with 4 mg/L BAP (86.66 ± 12.58% of explants regenerating shoots, and an average number of 5.45 ± 0.45 shoots per explant). Low percentages of In vitro rooting of regenerated shoots were observed with different concentrations of α-naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA). Nevertheless, the highest rooting percentage (13.33 ± 2.88%) was obtained on MS containing 2 mg/L NAA. These results provide new information for mass regeneration and the production of healthy seedlings, and can be applied in grapefruit improvement programs.